Atmospheric Pressure Ionization (API) Sources including Electrospray (ES), Atmospheric Pressure Chemical Ionization (APCI) and Inductively Coupled Plasma (ICP) ion sources interfaced to mass analyzers are typically operated with a single sample introduction probe. In mass spectrometric applications where internal standards are required, additional components can be added to the primary sample solution where the resulting mixture is delivered through one probe into the API source. The mixture of compounds in a single solution introduced through the same probe are ionized and mass analyzed. A known sample when mixed with an unknown sample can serve as an internal mass scale or quantitation calibration standard for the unknown components peaks appearing in the mass spectrum acquired in this manner. However, mixing a known compound calibration solution with an unknown sample solution can have undesired analytical consequences. The known and unknown solution components may effect one another in an unpredictable manner during the solution transport or ionization process. One component may react with another in solution or one or more components may suppress the ionization efficiency of other components during the ionization process. A solution with a known component mixture may be difficult to eliminate as a source of chemical contamination in a probe which is running a series of unknown samples at the trace component level. If it is desirable to deliver a known solution as a mixture through the sample introduction probe on an intermittent basis, the occasional sample introduction will be subject to the constraints of solution flow rates through the probe, efficiency of mixing solutions, dead volume losses and flushing of the probe to eliminate the known solution prior to the next analysis. The invention avoids performance and sample introduction problems encountered when mixing liquid samples prior to ionization in an API source, by conducting simultaneous mass analysis of two different solutions without the need to mix solutions in the same probe prior to analysis. One aspect of the invention is the configuration and simultaneous operation of multiple probes or multiple sprayers or nebulizers within a probe assembly through which different sample solutions can be introduced simultaneously into an API source during operation.
In one embodiment of the invention, multiple sample introduction means have been configured in Electrospray Atmospheric Pressure Ion sources which are interfaced to mass analyzers. At least two sample introduction Electrospray probes are operated simultaneously in an Electrospray ion source. At least one ES probe is supplied a sample which is different from the sample solution supplied to additional ES probes operating within the same ES source chamber. In this manner a calibration solution can be introduced through one ES probe while an unknown sample is introduced through another ES probe or second channel within the same ES probe assembly. Ions produced from both solutions via the simultaneous spraying of both ES probes blend or mix in the atmospheric pressure ES chamber background gas prior to entering the orifice into vacuum. The mixture of ions resulting from the solutions delivered from at least two ES probes is simultaneously mass to charge (m/z) analyzed resulting in a mass spectrum containing an internal standard for calibrating or tuning the mass analyzer. The internal calibration standard contained within the acquired mass spectrum is achieved without mixing known and unknown samples in solution. Simultaneous introduction of different samples through multiple ES probes also enables the study of mixed ion and molecule reactions at atmospheric pressure in the ES source chamber prior to introduction into vacuum. Each ES sample introduction probe assembly can be configured with nebulization gas and liquid layered flow. An internal calibration solution can be included in the layered flow or the primary flow of any given ES probe configured in the ES source chamber. The individual sample solution flows or nebulization gas flows to any combination of ES probes can be switched on or off during an analytical run without the need to reposition probes. In another aspect of the invention, an Atmospheric Pressure Chemical Ionization (APCI) source assembly can be configured with multiple inlet channels or probes. These multiple APCI inlet probes can include pneumatic nebulization and the solution and gas flow supplied to each inlet probe can be individually or simultaneously turned on or off. In both the ES and APCI sources, multiple probe sample solution ionization can be controlled without the need to reposition probes by switching voltages, controlling the nebulization gas flows or controlling the sample solution flows. Configurations of multiple sample introduction inlet probes can also be extended to a system that has a combination of both Electrospray and APCI ion production means in the same API chamber. Each ES or APCI sample inlet probe can include pneumatic or ultrasonic nebulization.
Configurations of Electrospray ion sources which include more than one sample introduction needle or nebulizer have been described in the literature. Kostianinen and Bruins, Proceedings of the 41st ASMS Conference on Mass Spectrometry, 744a, 1993, described the configuration and use of an assembly of multiple Electrospray inlet tips with and without pneumatic nebulization mounted in an Electrospray ion source. Each ES tip was supplied the same sample solution delivered from a single pump with a single solution source. The sample solution, delivered from a liquid chromatography pump, flowed into an assembly or array of one, two or four ES or pneumatic nebulization assisted ES sprayer tips in an attempt to improve ion signal intensity at higher liquid flow rates. In the arrangement reported, the solution flow to individual sprayer tips could not be turned on and off independently and different solutions could not be introduced selectively to individual sprayer tips in the assembly of multiple ES sprayer tips.
Rachel R. Ogorzalek Loo, Harold R. Udseth, and Richard D. Smith, Proceedings of the 39th ASMS Conference on Mass Spectrometry and Allied Topics, 266-267, 1991 and J. Phys. Chem., 6412-6415, 1991 and Richard D. Smith, Joseph A. Loo, Rachel R. Ogorzalek Loo, Mark Busman, and Harold R. Udseth, Mass Spectrometry Reviews, 10, 359-451,1991 describe the configuration of an Electrospray ion source interfaced to a quadruple mass analyzer apparatus which included dual Electrospray ion sources delivering ions to two separate entrance apertures of a Y shaped capillary. Positive ions created in one Electrospray source were introduced into one inlet branch of the Y shaped capillary and negative ions created from the second Electrospray ion source were introduced into the second inlet branch of the Y shaped capillary. The positive and negative ions swept into the two entrance orifices of the capillary tube began mixing where the two inlet branches of the capillary tube met well downstream of the capillary entrances located in the two ES atmospheric pressure source chambers. Dual Electrospray ionization sources or a separate ES source and a gas phase corona discharge source individually delivered ions into two entrance orifices of a Y shaped capillary. For all experiments reported, the first ES source produced ions of opposite polarity to the second ES or gas phase corona discharge source. The opposite polarity ions produced in separate ion sources were not mixed in the atmospheric pressure ion source but entered a split capillary tube at two separate entrance orifices and mixed in partial vacuum downstream in the capillary tube.
Bordoli, Woolfit and Bateman, Proceedings of the 43th ASMS Conference on Mass Spectrometry and Allied Topics, 98, 1995 described an Electrospray ion source which included a calibration ES probe configured with a second microtip (50 nl/min flow rate) sample probe interfaced to a magnetic sector mass analyzer. The sample probe included a microtip attached directly to a syringe needle. The syringe was mounted on an X-Y-Z positioning stage to optimize the position of the microtip sprayer. The calibration ES probe was configured such that it could be moved into a position when a calibration solution was sprayed at 500 nl/min while no sample flowed through the primary ES sample probe. After acquisition of a calibration mass spectrum, the calibration ES probe was retracted and the calibration solution flow turned off. The sample flow through the microtip sample ES probe was then turned on and a separate mass spectrum was acquired from the Electrosprayed ions produced. In this manner, an external calibration mass spectrum was acquired prior to acquisition of a mass spectrum of the primary sample. The calibration mass spectrum and the sample mass spectrum were then added together in the data system prior to calculating the mass assignment of the sample related peaks. For the ES source configuration reported, the two ES probes were not operated simultaneously and no gas phase mixture of calibration and sample ions was created at atmospheric pressure and no mass spectrum was acquired from a mixture of calibration and sample ions. No single mass spectrum was acquired which included sample related peaks and calibration compound related peaks with the apparatus described. Neither ES probe described was configured to operate with pneumatic nebulization assisted Electrospray. The ES calibration probe position required adjustment prior to acquiring a calibration spectrum to enable effective spaying near the orifice into vacuum. After acquisition of a calibration mass spectrum, the ES calibration probe was retracted to avoid interference prior to the mass spectrum acquisition from the sample solution delivered through the primary ES probe.
In one embodiment of the invention described, multiple samples are introduced into an API source simultaneously where ions are produced from all samples and mixed in the atmospheric pressure ion source chamber. A portion of the gas phase ion mixture is then swept into vacuum through an orifice or capillary where the ions are mass analyzed. In this manner a solution containing calibration compounds can be ionized simultaneously with a sample solution resulting in an acquired mass spectrum containing an internal standard without mixing calibration components and sample components in solution. Higher mass accuracy""s can be achieved with an internal standard when m/z assignments are calculated for sample ion related peaks in an acquired mass spectrum. In addition to independently introducing calibration compounds in an API source, multiple sample inlet probes can be used to introduce multiple samples individually or simultaneously into an API source. Mounting multiple probes in an API chamber such as ES and APCI probes, allows multiple ionization techniques to be run individually or simultaneously in a single API source assembly. Multiple Electrospray probes can be configured to collectively provide optimal performance over a wide range of sample flow rates and solution chemistries. ES probe positions can be configured to fall directly on the vacuum orifice centerline to a position angled to well over 100 degrees off the centerline. Different liquid flow rates can be delivered to separate ES or APCI probes within the same API source. ES and/or APCI probes mounted at different positions in the ES source chamber, can operate simultaneously, in pairs or in groups at different flow rates and introducing different sample solutions. The multiple ES probes may be operated with or without nebulization assist.
One embodiment of the invention is the configuration of an API source with multiple sample solution inlets, connected to different sample delivery systems, interfaced to a mass analyzer. Individual sample inlet probes can be operated independently or simultaneously in the same API source chamber. The composition and flow rate of solution introduced through each individual API probe can be controlled independently from other sample introduction ES, APCI or ICP probes. Multiple samples are introduced into the API source through multiple API probes without mixing separate sample components in solution prior to solution spraying and ionization. Ionization of components from multiple sample solutions occurs in the gas phase at or near atmospheric pressure. The API source may include but is not limited to Electrospray, APCI or ICP ionization means or combinations of each ionization technique. Another aspect of the invention is the technique of introducing a calibration solution into at least one API source inlet probe and the sample of interest through another API source inlet probe. Both calibration and sample solutions are introduced through separate inlet probes but are sprayed and ionized simultaneously in the API source resulting in a mixture of gas phase calibration and sample related ions. A portion of the resulting ion mixture is mass analyzed producing a mass spectrum which includes known component ion peaks that can serve as an internal standard to improve m/z measurement and even quantitation accuracy. Alternatively, multiple sample solutions can be introduced separately but simultaneously creating a mixture of ions at or near atmospheric pressure to study gas phase ion and molecule interactions and reactions. Multiple inlet probe API sources can be interfaced to any MS or MS/MSn mass analyzer type including but not limited to, Time-Of-Flight (TOF), Quadrupole, Fourier Transform (FTMS), Ion Trap, Magnetic Sector or a Hybrid mass analyzer.
In one embodiment of the invention, an Electrospray ion source is configured with multiple Electrospray probes. Each probe may or may not be configured with pneumatic or ultrasonic nebulization assist and/or a second liquid layer. The multiple ES probes and each liquid layer of each ES probe may be connected to different liquid delivery systems. In this manner, different samples, mixture of samples and/or solvents can be sprayed simultaneously or individually in a variety of combinations. The liquid delivery systems include but are not limited to liquid chromatography pumps, syringe pumps, gravity feed vessels, pressurized vessels, and or aspiration feed vessels. Samples may also be introduced using auto injectors, separation systems such as liquid chromatography (LC) or capillary electrophoresis (CE), capillary electrophoresis chromatography (CEC) and/or manual injection values connected to any or all ES probes. Multiple and independent solution introduction allows multiple samples to be analyzed simultaneously with Electrospray ionization without changing ES probe positions. The ability to introduce sample solution through one ES probe and have the option to selectively and simultaneously introduce additional secondary samples into the ES chamber through other ES probes can be used to generate mass spectra, even on-line during LC or CE separations, with internal or external calibration standards. Different sample mixtures which span a range of m/z values or sample types can be introduced through different ES probes. Depending on the unknown sample being analyzed, an optimal calibration solution can be chosen from another ES probe. For example one m/z range calibration solution can be chosen which produces singly charged ES ions when analyzing singly charged compounds and likewise multiple charged ES generated calibration ions can be produced when analyzing compounds which form multiply charged ions in Electrospray ionization. The solution flow for any secondary ES probe can be controlled independent of the solution flow to a primary ES sample solution probe without having to change or adjust any probe position, change the ES source voltages, shut off the primary sample solution flow or contaminate the solution being introduced through the primary sample solution probe. Multiple probe sets can be operated simultaneously or in sequence with other probe sets in the same API chamber. The configuration and operation of multiple ES probes can facilitate API MS detection from multiple sample sources. In particular, multiple sample probes facilitates and improves the analytical throughput of unattended automated operation of a single mass analyzer as a detector for multiple Liquid Chromatography separations systems.
In another embodiment of the invention, multiple nebulizers are configured in an Atmospheric Pressure Chemical Ionization source. Similar to ES, multiple sample solutions can be introduced into the gas phase and ionized without mixing solutions. In this APCI source embodiment, multiple nebulizers spray individual sample bearing solutions into a vaporizer where the mixture of nebulized droplets is evaporated prior to ionization in the corona discharge region. Calibration solutions can be introduced through one or more sample inlet probes independently and simultaneously with sample solution introduction through yet another inlet probe. No adjustment to probe position, applied voltages or vaporizer temperature may be required when controlling the solution flow to multiple inlet probes. This independent sample flow control with little or no mechanical adjustment in an APCI source increases the system level analytical flexibility and sample throughput with manual or automated operation while minimizing multiple solution cross contamination. Multiple APCI and ES probes can be configured in one API source in another embodiment of the invention. The combination ES and APCI source expands the range of analytical capability of an API-MS instrument interfaced to a variety of separation systems particularly for automated operation with a variety of samples.
The use of multiple probes with API sources, including ES, APCI or ICP ionization techniques allows a more rapid introduction of samples particularly when a fast mass analyzer such as Time-Of-Flight is used. Rapid sample introduction can be limited by the cycle time of an LC, CE or CEC separation system or auto injector. Sample introduction cycle time can also be limited by the time it takes for an injected sample to travel from the injector valve to the ES or APCI probe outlet. Multiple LC, CE or CEC, auto injectors, injector valves and API probes can be configured to decrease the cycle time of sample introduction and analysis time of an API MS system.